Methods using RANK/RANKL antagonist antibodies for treating pain

ABSTRACT

Disclosed herein are methods of treating pain using comprising RANK/RANKL antagonists.

CROSS-REFERENCE TO RELATED CASES

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/182,378, filed Jun. 14, 2016, now issued as U.S. Pat. No.9,493,541, which is a continuation of U.S. application Ser. No.14/952,724, filed Nov. 25, 2015, now issued as U.S. Pat. No. 9,371,392,which is a continuation of U.S. application Ser. No. 14/812,989, filedJul. 29, 2015, now issued as U.S. Pat. No. 9,205,045, which is acontinuation of U.S. patent application Ser. No. 14/495,732, filed Sep.24, 2014, now issued as U.S. Pat. No. 9,127,069, which claims priorityto U.S. Provisional Patent Application Nos. 62/012,112, filed Jun. 13,2014 and 62/010,754, filed Jun. 11, 2014.The entire disclosures of theseapplications are incorporated herein by reference.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing incomputer readable format. The Sequence Listing is provided as a fileentitled 1958603-00179_ST25_SEQUENCE_LISTING.txt, created Sep. 1, 2016,which is 14,833 bytes in size. The information in the computer readableformat of the sequence listing is incorporated herein by reference inits entirety.

BACKGROUND

The receptor activator of nuclear factor KB (RANK), receptor activatorof nuclear factor KB ligand (RANKL), and osteoprotegerin (OPG) triad(RANK/RANKL/OPG) play an important role in immune response and bonemetabolism. RANK/RANKL triggers a network of TRAF-mediated kinasecascades that promote osteoclast differentiation. RANKL is expressed onosteoblast cells and its receptor, RANK, on pre-osteoclastic cells. TheRANK/RANKL interaction induces the differentiation and formation ofmultinucleated mature osteoclasts, causing bone resorption. The thirdprotagonist, OPG, is also produced by osteoblasts and exerts aninhibitory effect on the pre-osteoclastic differentiation process. OPG,by binding to RANKL, inhibits the RANK/RANKL interaction and subsequentosteoclastogenesis.

SUMMARY

Described herein are dosage forms comprising RANK/RANKL antagonists fortreatment of various diseases or medical conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the mean paw compression thresholds of ratstreated with vehicle or RANK/RANKL antagonism over time in a rat modelof inflammatory pain.

FIG. 2 illustrates the Total Pain Relief over time for rats treated withvehicle or RANK/RANKL antagonism in a rat model of inflammatory pain.

FIG. 3 illustrates the percent reversal of arthritis pain with differentdoses of RANK/RANKL antagonism in a rat model of arthritis pain.

FIG. 4 depicts the mean pain threshold of rats treated with vehicle orRANK/RANKL antagonism in a rat model of arthritis pain.

FIG. 5 illustrates the reversal of CRPS pain with RANK/RANKL antagonism.

DETAILED DESCRIPTION

Provided herein are methods and compositions for treating undesirableconditions or diseases, including pain. Generally, the patient is ahuman, but the methods described herein may be applied to any mammal,including domestic animals such as pets and farm animals. Typically, apatient in need of treatment receives an amount of a RANK/RANKLantagonist that is effective to treat pain or another undesirablecondition.

Some embodiments relate to the treatment of pain or other undesirableconditions by blocking RANK/RANKL function. For example, an effectiveamount of one or more RANK/RANKL antagonists can be administered to amammal, such as a human being, for the treatment of pain or anotherundesirable condition.

A RANK/RANKL antagonist can be any chemical species having the activityof RANK/RANKL antagonists as commonly understood in the art, or can bean agent that partially or fully blocks, inhibits, or neutralizes one ormore biological activities of RANKL or RANK, such as binding of RANKL toRANK, in vitro, in situ, or in vivo. An antagonist may function in adirect or indirect manner. For instance, the antagonist may directlybind to RANKL or RANK, thus partially or fully blocking, inhibiting orneutralizing one or more biological activities of RANKL or RANK, invitro, in situ, or in vivo. The antagonist may also function indirectlyto partially or fully block, inhibit or neutralize one or morebiological activities of RANKL or RANK, in vitro, in situ, or in vivo asa result of interacting with, e.g., activating, inducing, blocking orinhibiting, another compound that can bind to RANK or RANKL. Theantagonist may also function indirectly to partially or fully block,inhibit or neutralize one or more biological activities of RANKL orRANK, in vitro, in situ, or in vivo as a result of modulating oraffecting the production of RANKL or RANK.

“Treatment,” “treating,” or “therapy” includes its common meaning in thefield and includes diagnosis, cure, mitigation, treatment, andprevention.

A RANK/RANKL antagonist can comprise a biomolecule, such as apolypeptide or a protein, a nucleic acid, a polysaccharide, etc., orcould be a small molecule, such as a compound having a molecular weightthat is less than about 2000, about 1000, about 500, etc. “Polypeptide”as used herein includes its common meaning in the field as well asmolecules derived from two amino acids or more.

A RANK/RANKL antagonist can be administered with one or more additionaltherapeutically active agents. For example, a RANK/RANKL antagonist canbe administered simultaneously or administered consecutively with asecond therapeutically active agent.

There are a variety of polypeptides and proteins that can be suitableRANK/RANKL antagonists. In some embodiments, the RANK/RANKL antagonistcan be a protein that can be derived from the same species of animal asthe patient or subject.

In some embodiments, patients in need thereof can treated byadministering a RANK/RANKL antagonist comprising a soluble RANK proteinthat is capable of binding RANKL that can comprise all or a fragment ofthe extracellular domain of a RANK protein. The patient can be a humanand the soluble RANK can be derived from a human RANK polypeptide.RANK/RANKL antagonists comprising a soluble RANK polypeptide may includeother portions of RANK besides the extracellular domain but do notinclude the transmembrane region.

In some embodiments, fusion proteins comprising a soluble RANK for useas antagonists suitable for the methods described herein are thosedescribed in US 2003/0021785 A1, which is incorporated here in full byreference.

In some embodiments, soluble RANK polypeptides suitable for the methodsdescribed herein can be those described in US 2003/0021785 A1, which isincorporated here in full by reference.

In some embodiments, soluble RANK proteins for use as antagonistssuitable for the methods described herein are those described in US2003/0021785 A1, which is incorporated here in full by reference.

The terms “OPG” or “osteoprotegerin” or “OPG receptor” include theircommon meaning in the field and includes “native sequence OPGpolypeptides” and “OPG variants” (which are further defined herein).“OPG” can be a designation given to those polypeptides which can beencoded by the nucleic acid molecules comprising the polynucleotidesequences shown in Simonet et al., Cell, 89:309 (1997) and variantsthereof, as well as fragments of any of the sequences referred to above.The OPG polypeptides may be isolated from a variety of sources, such asfrom human tissue or cells or from another source, or prepared byrecombinant and/or synthetic methods. A “native sequence” OPGpolypeptide includes its common meaning in the field and additionallyincludes the following: comprises a polypeptide having the same aminoacid sequence as the corresponding OPG polypeptide derived from nature.Such native sequence OPG polypeptides can be isolated from nature or canbe produced by recombinant and/or synthetic means. The term “nativesequence OPG polypeptide” includes its common meaning in the field andadditionally includes the following: specifically encompassesnaturally-occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of thepolypeptide. The OPG polypeptides include the polypeptides described as“FDCR-1” and “OCIF” in Yasuda et al., Endocrinology, 139:1329 (1998) andYun et al., J. Immunol., 1 61:61 13-6121 (1998).

“OPG variant” includes its common meaning in the field and includes anOPG polypeptide having at least about 80% amino acid sequence identitywith the amino acid sequence of a native sequence OPG or OPG ECD. Insome embodiments, the OPG variant binds to RANKL, such as, to the fulllength RANK Ligand.

An “extracellular domain” or “ECD” include their common meaning in thefield and include: a form of the polypeptide which is essentially freeof the transmembrane and cytoplasmic domains. Ordinarily, an ECD form ofa polypeptide will have less than about 1% of such transmembrane and/orcytoplasmic domains and possibly, will have less than about 0.5% of suchdomains. It can be understood that any transmembrane domain(s)identified for the polypeptides can be identified pursuant to criteriaroutinely employed in the art for identifying that type of hydrophobicdomain. The exact boundaries of a transmembrane domain may vary. In someembodiments, the boundaries of a transmembrane domain varies by no morethan about 5 amino acids at either end of the domain as initiallyidentified. In some embodiments, the ECD will consist of a soluble,extracellular domain sequence of the polypeptide which is free of thetransmembrane and cytoplasmic or intracellular domains (and is notmembrane bound).

A “liposome” includes its common meaning in the field and includes: asmall vesicle composed of various types of lipids, phospholipids and/orsurfactant which can be useful for delivery of a drug (such as apolypeptide or antibody thereto) to a mammal. The components of theliposome can be commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

The term “antibody” includes its common meaning in the field andincludes use in the broadest sense, and additionally, specificallycovers, for example, single monoclonal antibodies which can bind RANKLor RANK, antibody compositions with polyepitopic specificity, singlechain antibodies, and fragments of antibodies.

Some RANK/RANKL antagonists may comprise an antibody, such as amonoclonal antibody.

The term “monoclonal antibody” as used herein includes its commonmeaning in the field and includes: an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population can be identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies can be highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody can be directed against a single determinant onthe antigen. In addition to their specificity, the monoclonal antibodiescan be advantageous in that they can be synthesized by hybridomaculture, uncontaminated by other immunoglobulins. The modifier“monoclonal” includes its common meaning in the field and indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies may be made by the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581 -597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) which includes its common meaning in thefield and includes: a portion of the heavy and/or light chain can beidentical to or homologous with corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) can be identicalto or homologous with corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851 -6855 (1984)). Methods of makingchimeric antibodies are known in the art.

“Humanized” forms of non-human (e.g., murine) antibodies can be chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies can be human immunoglobulins(recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient can bereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity,and capacity. In some instances, Fv framework region (FR) residues ofthe human immunoglobulin can be replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues whichcan be found neither in the recipient antibody nor in the imported CDRor framework sequences. These modifications can be made to furtherrefine and maximize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions can be those of a humanimmunoglobulin sequence. The humanized antibody may also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details, see Jones et al.,Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). Thehumanized antibody may be an antibody wherein the antigen-binding regionof the antibody can be derived from an antibody produced by immunizingmacaque monkeys with the antigen of interest. Methods of makinghumanized antibodies are known in the art.

Human antibodies can also be produced using various techniques known inthe art, including phage-display libraries. Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies. Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147(1):86-95 (1991).

“Antibody fragments” comprise a portion of an intact antibody, such asthe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, a designation reflecting the ability to crystallizereadily. Pepsin treatment yields an F(ab′)₂ fragment that has twoantigen-combining sites and can be still capable of cross-linkingantigen.

“Fv” can be the minimum antibody fragment which contains a completeantigen-recognition and binding site. This region consists of a dimer ofone heavy- and one light-chain variable domain in tight, non-covalentassociation. It can be in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. The Fab fragment alsocontains the constant domain of the light chain and the first constantdomain (CH1) of the heavy chain. Fab fragments differ from Fab′fragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five known major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. The Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which can enable the sFv toform the desired structure for antigen binding. For a review of sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” includes its common meaning in the field andincludes: small antibody fragments with two antigen-binding sites, whichfragments comprise a heavy-chain variable domain (V_(H)) connected to alight-chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains can be forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/011161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993). An antibody that “specificallybinds to” or can be “specific for” a particular polypeptide or anepitope on a particular polypeptide can be one that binds to thatparticular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.

The term “biosimilar” or “biosimilarity,” in reference to a biologicalproduct, means that the biological product is highly similar to thereference product notwithstanding minor differences in clinicallyinactive components, and there are no clinically meaningful differencesbetween the biological product and the reference product in terms of thesafety, purity, and potency of the product.

The term “interchangeable” or “interchangeability,” in reference to abiological product, means that the biological product may be substitutedfor the reference product without the intervention of a health careprovider who prescribed the reference product. An interchangeablebiological product is biosimilar to a reference product, and can beexpected to produce the same clinical result as the reference product inany given patient.

The term “reference product” means the single biological product againstwhich a biological product is evaluated. In some embodiments, thereference product may be an FDA-licensed biological product.

The term “biological product” means a virus, therapeutic serum, toxin,antitoxin, vaccine, blood, blood component or derivative, allergenicproduct, protein (except any chemically synthesized polypeptide), oranalogous product, or arsphenamine or derivative of arsphenamine (or anyother trivalent organic arsenic compound), applicable to the prevention,treatment, or cure of a disease or condition. In some embodiments, thedisease or condition is a disease or condition of human beings.

In some embodiments, the RANKL antagonist can be an OPG(osteoprotegerin) variant or an anti-RANKL antibody; the RANKLantagonist can be a monoclonal anti-RANKL antibody; the RANKL antagonistcan be a humanized monoclonal anti-RANKL antibody; the RANKL antagonistcan be denosumab, or a biosimilar thereof; the RANKL antagonist can bean antibody disclosed herein, or a biosimilar thereof; or the RANKLantagonist can be OPG.

In some embodiments, the RANK/RANKL antagonist can be denosumab.Denosumab is a fully human antibody that shares the pharmacologicalattributes of OPG, in that both bind to and inhibit RANKL. Additionally,denosumab can have a significant longer half-life than OPG, allowingless frequent administration. In some embodiments, the RANK/RANKLantagonist can be OPG. Further description of denosumab may be found inWO 2013/181575 and U.S. Pat. Nos. 6,740,522; 7,097,834; 7,364,736;7,411,050; 7,744,886; 7,923,008; 8,058,418; 8,333,963; 8,377,690; and8,409,578, each of which are incorporated here in full by reference.

In some embodiments, the RANK/RANKL antagonist is a biosimilar ofdenosumab.

In some embodiments, the RANK/RANKL antagonist is a biological productthat is interchangeable with denosumab.

In some embodiments, the RANK/RANKL antagonist can be an OPG(osteoprotegerin) variant or an anti-RANKL antibody. In someembodiments, the RANK/RANKL antagonist can be a monoclonal anti-RANKLantibody. In some embodiments, the RANK/RANKL antagonist can be a smallinterfering RNA, a microRNA, a precursor molecule, a ribozyme, anantisense nucleic acid sequence, or an aptamer targeting RANKL. In someembodiments, the RANK/RANKL antagonist can be a humanized monoclonalanti-RANKL antibody, or the RANK/RANKL antagonist can be a humanizedmonoclonal anti-RANKL antibody which competes with denosumab for bindingto RANKL.

Also disclosed herein are antibodies or antibody fragments which competefor a binding epitope on human RANKL with denosumab and thus bind tosubstantially the same epitope on RANKL as denosumab. In certainembodiments, the antibody is a humanized antibody or a chimericantibody. Also disclosed herein are methods of using such antibodies totreat a human subject suffering from pain mediated by RANK/RANKL.

In some embodiments, the antibody which competes for binding withdenosumab to RANKL is a chimeric monoclonal antibody comprising thecomplementarity determining regions (CDRs) of denosumab fused to humanimmunoglobulin framework (FR) and constant (C) regions.

In some embodiments, the antibody that competes for binding withdenosumab to RANKL is a biosimilar of denosumab. In some embodiments,the antibody that competes for binding with denosumab to RANKL is abiological product that is interchangeable with denosumab.

The substitution of denosumab CDRs into any human variable domain FR maynot allow the same spatial orientation provided by the conformation tothe parent variable FR from which the CDRs originated. The heavy andlight chain variable framework regions to be paired in the finalantibody can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies, be derived from human germlineimmunoglobulin sequences, or can be consensus sequences of several humanantibody and/or germline sequences.

Suitable human antibody sequences can be identified by computercomparisons of the amino acid sequences of the mouse variable regionswith the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each.

Humanized antibodies can be isolated from a library of variant sequencesthat can be screened for the desired activity, binding affinity, orspecificity. One format for creation of such a library of variants is aphage display vector. Alternatively, variants can be generated usingother methods for variegation of a nucleic acid sequence encoding thetargeted residues within the variable domain.

A method of determining whether further substitutions are required tooptimize affinity in a humanized or chimeric antibody, and the selectionof amino acid residues for substitution, can be accomplished usingcomputer modeling. Computer hardware and software for producingthree-dimensional images of immunoglobulin molecules are widelyavailable. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model. Thesolved starting structures are modified to allow for differences betweenthe actual amino acids in the immunoglobulin chains or domains beingmodeled, and those in the starting structure. The modified structuresare then assembled into a composite immunoglobulin. Finally, the modelis refined by energy minimization and by verifying that all atoms arewithin appropriate distances from one another and that bond lengths andangles are within chemically acceptable limits.

Because of the degeneracy of the code, a variety of nucleic acidsequences can encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de novo solid-phaseDNA synthesis or by PCR mutagenesis of an earlier prepared variant ofthe desired polynucleotide.

The variable segments of humanized and chimeric antibodies produced asdescribed herein are typically linked to at least a portion of a humanimmunoglobulin constant region. The antibody will contain both lightchain and heavy chain constant regions. The heavy chain constant regionusually includes CHI, hinge, CH2, CH3, and, sometimes, CH4 domains.

The humanized and chimeric antibodies may comprise any type of constantdomains from any class of antibody, including IgM, IgG, IgD, IgA andIgE, and any subclass (isotype), including IgG1, IgG2, IgG3 and IgG4. Ifit is desired that the humanized or chimeric antibody exhibit cytotoxicactivity, the constant domain is usually a complement-fixing constantdomain and the class is typically IgG1. When such cytotoxic activity isnot desirable, the constant domain may be of the IgG2 class. Thehumanized or chimeric antibody may comprise sequences from more than oneclass or isotype.

Nucleic acids encoding humanized or chimeric light and heavy chainvariable regions, optionally linked to constant regions, are insertedinto expression vectors. The light and heavy chains can be cloned in thesame or different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Such control sequences include a signal sequence, apromoter, an enhancer, and a transcription termination sequence.

Once a chimeric or humanized antibody molecule has been identifiedaccording to the structural and functional characteristics describedherein, nucleic acid sequences encoding the desired portions of, or theentire antibody chains, can be cloned, copied, or chemically synthesizedand can be isolated and used to express the antibody by routine methods.The humanized or chimeric antibody may be purified by any method knownin the art for purification of an immunoglobulin molecule, for example,by chromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. In addition,the humanized or chimeric antibodies, or fragments thereof, can be fusedto heterologous polypeptide sequences described herein or otherwiseknown in the art, to facilitate purification.

In some embodiments, the nucleic acid molecules encoding a soluble RANKfor use as a RANK/RANKL antagonist suitable for the methods describedherein are those described in US 2003/0021785 A1, which is incorporatedhere in full by reference.

In some embodiments, antisense RNA and DNA molecules for use asantagonists suitable for the methods described herein are thosedescribed in US 2003/0021785 A1, which is incorporated here in full byreference.

Small interfering RNA (short interfering RNA, silencing RNA, siRNA) canbe a class of double-stranded RNA-molecules, which can be 19-30nucleotides, such as 20-25 nucleotides long. siRNAs inhibit expressionof a specific gene via RNA-interference. siRNAs can be cut from longdouble-stranded RNAs by the RNase III Dicer. They can also be derived bychemical synthesis. They also play a role in antiviral mechanisms or inshaping the chromatin structure of a genome. In molecular research,synthetic siRNAs can also be used in RNA-interference (RNAi) todownregulate the expression of specific target genes. With their abilityto knock down essentially any gene of interest, siRNAs can been used toknock down RANK or RANKL. MicroRNAs (miRNAs) can be posttranscriptionalregulators that bind to complementary sequences in the 3′UTR of mRNAtranscripts, usually resulting in gene silencing. They can be short RNAmolecules which can be about 22 nucleotides long.

Precursor molecules, e.g. precursor molecules of siRNA and/or miRNA, maybe substrates for the siRNA/miRNA-biogenesis-apparatus of the targetcell. This comprises, for example, RNA precursor molecules such asdouble-stranded RNA (dsRNA) or short hairpin RNA-molecules (shRNA),which can be processed by endonucleases such as Drosha and/or Pasha tosiRNA molecules or miRNA molecules, respectively. In some embodiments,dsRNA-molecules or short hairpin RNA-molecules (shRNA) having a lengthof more than 27 nucleotides, more than 30 up to 100 nucleotides orlonger, or dsRNA molecules having a length of 30-50 nucleotides, can beused.

Further precursor molecules may be DNA constructs encoding dsRNA, shRNA,siRNA and/or miRNA, whereby the coding elements can be controlled byregulatory elements allowing an expression of dsRNA, shRNA, siRNA,and/or miRNA in the target cell. Examples for such control elements canbe polymerase II or promoters or polymerase III promoters such as, forexample, U6 or H1. Ribozymes can be catalytic RNAs which possess a welldefined structure that enables them to catalyze a chemical reaction.Apart from naturally occurring ribozymes they can be made artificiallyand be tailored to interact with nucleic acids and proteins.

Antisense oligonucleotides can be single strands of DNA or RNA that canbe complementary to a chosen sequence. They can be between 10 and 35nucleotides long, such as about 20-25 nucleotides. Antisense DNAoligonucleotides can target specific, complementary RNA, and uponbinding DNA/RNA hybrids can be formed. Antisense RNA oligonucleotidescan bind to mRNA by binding to mRNA strands.

Aptamers can be oligonucleic acid (DNA or RNA aptamers) or peptidemolecules (peptide aptamers) that bind to a specific target molecule.Aptamers can be used for therapeutic purposes as macromolecular drugs.Aptamers can be created by selecting them from a large random sequencepool.

A RANK/RANKL antagonist can be a small molecule, e.g. a compound that isnot a biomolecule, such as a compound having a molecular weight belowabout 2000, Daltons, 1000 Daltons, or 500 Daltons.

Some Bruton's tyrosine kinase (BTK) inhibitors can be RANK/RANKLantagonists. BTK is primarily expressed in B cells, myeloid anderythroid progenitor cells.

BTK inhibitors can include ONO-4059; ibrutinib;Benzo[b]thiophene-2-carboxamide,N-[3-[6-[[4-[(2R)-1,4-dimethyl-3-oxo-2-piperazinyl]phenyl]amino]-4,5-dihydro-4-methyl-5-oxo-2-pyrazinyl]-2-methylphenyl]-4,5,6,7-tetrahydro-(GDC-0834);RN-486; Benzamide,4-(1,1-dimethylethyl)-N-[3-[8-(phenylamino)imidazo[1,2-a]pyrazin-6-yl]phenyl]-(CGI-560);Benzamide,N-[3-[4,5-dihydro-4-methyl-6-[[4-(4-morpholinylcarbonyl)phenyl]amino]-5-oxo-2-pyrazinyl]-2-methylphenyl]-4-(1,1-dimethylethyl)-(CGI-1746CASRegistry No. 910232-84-7); HM-71224; 2-Propenamide,N-[3-[[5-fluoro-2-[[4-(2-methoxyethoxy)phenyl]amino]-4-pyrimidinyl]amino]phenyl]-(CC-292,CAS Registry No. 1202757-89-8); 2-Pyridinecarboxamide,4-[4-[[5-fluoro-4-[[3-[(1-oxo-2-propen-1-yl)amino]phenyl]amino]-2-pyrimidinyl]amino]phenoxy]-N-methyl-(CNX-774,CAS Registry No. 1202759-32-7), AVL-101 (CAS Registry No. 1552307-34-2),AVL-291 (CAS Registry No. 1552307-35-3), and AVL-292 (CAS Registry No.1552307-36-4), [N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide](dasatinib), alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-ibromophenyl)propenamide (LFM-A13), and ONO-WG-307.

ONO-4059 has been shown to dose-dependently inhibit RANKL-drivenosteoclast differentiation by 70% (IC₅₀: 0.853 nmol/L) (Ariza, Yuko,Yoshizawa, Toshio, Ueda, Yoshiko, Hotta, Shingo, Narita, Masami,Kawabata, Kazuhito; ONO-4059—A Novel Small Molecule Bruton's TyrosineKinase (Btk) Inhibitor, Suppresses Osteoclast Differentiation andActivation. [abstract]. Arthritis Rheum 2012;64 Suppl 10 :1799)

Compounds such as pamidronate or pamidronic acid, incadronate orincadronic acid, ibandronate or ibandronic acid, risedronate orrisedronic acid, zoledronate or zoledronic acid, minodronate orminodronic acid, cimadronate or cimadronic acid, etc., may be RANK/RANKLantagonists.

Zoledronic acid has been shown to “markedly increased OPG proteinsecretion and reduced transmembrane RANKL protein expression” (Pan etal. 2004, J. Bone Miner. Res. 2004 January; 19(1): 147-154).

Minodronic acid has been shown to “inhibit[] RANKL expression in acultured bone marrow stromal cell line.” (Nishida, et al., Biochemicaland Biophysical Research Communications 328 (2005) 91-97).

In some embodiments, the RANK/RANKL antagonist is zoledronic acid.

In some embodiments, the RANK/RANKL antagonist is minodronic acid.

In some embodiments, the RANKL antagonist is zoledronic acid.

In some embodiments, the RANKL antagonist is minodronic acid.

Some RANK/RANKL antagonists are RANKL antagonists as understood in theart, or include any molecule that partially or fully blocks, inhibits,or neutralizes a biological activity of RANKL. This includes, but is notlimited to, soluble forms of OPG or RANK, such as an extracellulardomain sequence of RANK, OPG immunoadhesins, RANK immunoadhesins, OPGfusion proteins, RANK fusion proteins, covalently modified forms of OPG,covalently modified forms of RANK, OPG variants, RANK variants, OPGantibodies, RANK antibodies, and RANKL antibodies.

Some RANK/RANKL antagonists are RANK antagonists as understood in theart, or include any molecule that partially or fully blocks, inhibits,or neutralizes a biological activity of RANK.

Unless otherwise indicated, any reference to a RANK/RANKL antagonists bystructure, name, or any other means, includes pharmaceuticallyacceptable salts, such as sodium, potassium, and ammonium salts;prodrugs, such as ester prodrugs; alternate solid forms, such aspolymorphs, solvates, hydrates, etc.; tautomers; or any other chemicalspecies that may rapidly convert to a compound described herein underconditions in which the compounds are used as described herein.

If stereochemistry is not indicated, a name or structural depictionincludes any stereoisomer or any mixture of stereoisomers.

Generally, an oral dosage form comprising a small molecule RANK/RANKLantagonist such as ibrutinib, zoledronic acid, or minodronic acid, canbe administered orally to a mammal, such as a human being, at leastonce, to treat a disease or condition, or to relieve pain

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may be used to treat, or provide relief of, any type ofpain including, but not limited to, back pain, pain in an extremity,arthralgia, muscle pain or myalgia, inflammatory pain, arthritis pain,complex regional pain syndrome, lumbosacral pain, musculoskeletal pain,neuropathic pain, chronic pain, cancer-related pain, acute pain,postoperative pain, rheumatoid arthritis, osteoarthritis, erosiveosteoarthritis, axial spondyloarthritis including ankylosingspondylitis, acute vertebral crush fracture, fibrous dysplasia, SAPHOsyndrome, osteoporosis, transient osteoporosis, or transientosteoporosis of the hip etc. In some embodiments, a RANK/RANKLantagonist, such as an antibody (e.g., denosumab, a biological productthat is interchangeable with denosumab, a biosimilar of denosumab, or anantibody which competes with denosumab for binding to RANKL), or a smallmolecule such as ibrutinib, zoledronic acid, or minodronic acid, may beused to treat, or provide relief of, any type of pain including, but notlimited to, inflammatory pain, arthritis pain, complex regional painsyndrome, lumbosacral pain, musculoskeletal pain, neuropathic pain,chronic pain, cancer-related pain, acute pain, postoperative pain,fibromyalgia, etc. In some instances, pain relief may be palliative, orpain relief may be provided independent of improvement of the disease orcondition or the underlying cause of the disease or condition. Forexample, although the underlying disease may not improve, or maycontinue to progress, an individual suffering from the disease mayexperience pain relief. In some embodiments, enhanced bioavailability ofthe zoledronic acid or minodronic acid may be achieved in treating oneof these conditions by administering a dosage form comprising zoledronicacid in the form of a disodium salt. This may allow a reduced molaramount of the disodium salt to be used as compared to what would be usedwith the diacid form.

In some embodiments, the RANK/RANKL antagonist, such as an antibody(e.g., denosumab, a biological product that is interchangeable withdenosumab, a biosimilar of denosumab, or an antibody which competes withdenosumab for binding to RANKL), or a small molecule such as ibrutinib,zoledronic acid, or minodronic acid, may be administered to relieveinflammatory pain including musculoskeletal pain, arthritis pain, andcomplex regional pain syndrome.

In some embodiments, a RANK/RANKL antagonist, such as an antibody (e.g.,denosumab, a biological product that is interchangeable with denosumab,a biosimilar of denosumab, or an antibody which competes with denosumabfor binding to RANKL), or a small molecule such as ibrutinib, zoledronicacid, or minodronic acid, may be administered orally to relieveinflammatory pain including musculoskeletal pain, arthritis pain, andcomplex regional pain syndrome.

In some embodiments, a RANK/RANKL antagonist that is an antibody orfragment thereof described herein has, respective of their correspondingregion (e.g., heavy chain, light chain, variable region, etc.) todenosumab, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95% or about 100% sequenceidentity to denosumab or a corresponding fragment thereof. In someembodiments, an antibody or fragment thereof described herein has about70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to denosumab or a correspondingfragment thereof. In some embodiments, an antibody or fragment thereofdescribed herein has about 95% or 99% sequence identity to denosumab ora corresponding fragment thereof.

In some embodiments, a RANK/RANKL antagonist that is an antibody orfragment thereof described herein has about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95% or about 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:1 and SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:5 and SEQ ID NO:6, or a corresponding fragment thereof.In some embodiments, a RANK/RANKL antagonist that is an antibody orfragment thereof described herein has about 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:1 and SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:5 and SEQ IDNO:6, or a corresponding fragment thereof. In some embodiments, aRANK/RANKL antagonist that is an antibody or fragment thereof describedherein has about 95% or 99% sequence identity to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or a correspondingfragment thereof. In some embodiments, a RANK/RANKL antagonist that isan antibody or fragment thereof described herein has about 95% or 99%sequence identity to SEQ SEQ ID NO:1 and about 95% or 99% sequenceidentity to SEQ ID NO:2, or about 95% or 99% sequence identity to SEQ IDNO:5 and about 95% or 99% sequence identity to SEQ ID NO:6. In someembodiments, a RANK/RANKL antagonist that is an antibody or fragmentthereof described herein has a) a heavy chain comprising CDR1, CDR2, andCDR3 of SEQ ID NO: 3; and b) a light chain comprising CDR1, CDR2, andCDR3 of SEQ ID NO: 4.

With respect to use of a RANK/RANKL antagonist, such as an antibody(e.g., denosumab, a biological product that is interchangeable withdenosumab, a biosimilar of denosumab, or an antibody which competes withdenosumab for binding to RANKL), or a small molecule such as ibrutinib,zoledronic acid, or minodronic acid, for relieving pain associated withan inflammatory condition or musculoskeletal pain, relief of pain can beshort-term, e.g. for a period of hours after administration of thedosage form, and/or relief of pain can be long-term, e.g. lasting fordays, weeks, or even months after administration of the RANK/RANKLantagonist. In some embodiments, a mammal, such as a human being,experiences pain relief at least about 3 hours, at least about 6 hours,at least about 12 hours, at least about 24 hours, at least about 48hours, at least about one week, at least about 2 weeks, or at leastabout 3 weeks, at least about 1 month, at least about 2 months, at leastabout 3 months, at least about 6 months, after administration ofdenosumab, minodronic acid, or oral zoledronic acid. In someembodiments, a mammal, such as a human being, experiences pain reliefduring at least part of the time from about 3 hours to about 2 weeks,about 3 hours to about 3 weeks, about 3 hours to about 24 hours, about 6hours to about 2 weeks, or about 6 hours to about 24 hours, about 3 daysto about 2 weeks, about 6 days to about 2 weeks, at least about 1 month,at least about 2 months, at least about 3 months, at least about 6months, after administration of denosumab, minodronic acid, or oralzoledronic acid.

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may be administered to relieve musculoskeletal painincluding back pain (such as low back pain), and pain associated withrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,erosive osteoarthritis, sero-negative (non-rheumatoid) arthropathies,non-articular rheumatism, peri-articular disorders, axialspondyloarthritis including ankylosing spondylitis, Paget's disease ofbone, fibrous dysplasia, SAPHO syndrome, transient osteoarthritis of thehip, vertebral crush fractures, osteoporosis, etc.

Examples of musculoskeletal pain include back pain (such as low backpain); and pain associated with vertebral crush fractures, fibrousdysplasia, osteogenesis imperfecta, Paget's disease of bone, transientosteoporosis, and transient osteoporosis of the hip.

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may also be used to treat bone fractures or to enhancethe healing of bone fractures.

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may also be used to treat low back pain, or othermusculoskeletal or inflammatory conditions, having a change in bone thatis detectable by MRI or another medical imaging instrument. For example,a RANK/RANKL antagonist may be used to treat low back pain associatedModic changes, or vertebral endplate signal changes (VESC) and bonemarrow changes visible using magnetic resonance imaging (MRI). Modicchanges, can be classified into various types including type 1 (M1),type 2 (M2), and type 3 (M3) lesions or changes, any of which may betreated using a RANK/RANKL antagonist. VESCs may be found in patientswith different types of low back pain including but not limited tospondylitis, trauma, spondyloarthropathies including ankylosingspondylitis, Schmorl's nodes, fracture, tumor, and spinal cordinfarction. Lesions in ankylosing spondylitis include osteitis andspondylodiscitis which can be detected using MRI or another medicalimaging instrument. Treatment with a RANK/RANKL antagonist, such as anantibody (e.g. denosumab, a biological product that is interchangeablewith denosumab, a biosimilar of denosumab, or an antibody which competeswith denosumab for binding to RANKL) or an oral or intravenous smallmolecule (such as ibrutinib, zoledronic acid, or minodronic acid) mayreduce the size of, or prevent the growth or progression of changes inbone that are detectable on MRI (e.g. Modic changes, VESC, bone marrowchanges, bone marrow edema, etc.).

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may also be used to treat arthritis. Arthritis refersto inflammatory joint diseases that can be associated with pain.Examples of arthritis pain include pain associated with osteoarthritis,erosive osteoarthritis, rheumatoid arthritis, juvenile rheumatoidarthritis, psoriatic arthritis, sero-negative (non-rheumatoid)arthropathies, non-articular rheumatism, peri-articular disorders,neuropathic arthropathies including Charcot's foot, axialspondyloarthritis including ankylosing spondylitis, and SAPHO syndrome.

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may also be used to treat arthritis, such asosteoarthritis of the knee, including arthritis or osteoarthritis of theknee associated with bone marrow lesions (BML), including BML that maybe detected using MRI or another medical imaging instrument. In someembodiments, a RANK/RANKL antagonist may be used to treat arthritis orosteoarthritis of the knee associated with bone marrow edema (BM E),including BME which may be detected using MRI or another medical imaginginstrument.

In some embodiments, a human being that is treated for a disease orcondition, such as an inflammatory condition, e.g. arthritis, with aRANK/RANKL antagonist, has an age of about 10 years to about 90 years,about 20 years to about 80 years, about 30 years to about 75 years,about 40 years to about 70 years, about 1 year to about 16 years, orabout 80 years to about 95 years.

In some embodiments, a human being that is treated for a disease orcondition, such as an inflammatory condition or musculoskeletal pain,e.g. arthritis, with a RANK/RANKL antagonist, has suffered from thedisease or condition for at least 1 month, at least 2 months, at least 6months, or at least 1 year.

In some embodiments, the disease or condition, such as an inflammatorycondition, e.g. arthritis, affects a knee, an elbow, a finger, a toe, awrist, a shoulder, or a hip.

In some embodiments, the RANK/RANKL antagonist, such as an antibody(e.g., denosumab, a biological product that is interchangeable withdenosumab, a biosimilar of denosumab, or an antibody which competes withdenosumab for binding to RANKL), or a small molecule such as ibrutinib,zoledronic acid, or minodronic acid, may also be administered to relieveneuropathic pain, including diabetic peripheral neuropathy,post-herpetic neuralgia, trigeminal neuralgia, monoradiculopathies,phantom limb pain, and central pain. Other causes of neuropathic paininclude cancer-related pain, lumbar nerve root compression, spinal cordinjury, post-stroke pain, central multiple sclerosis pain,HIV-associated neuropathy, and radio-therapy or chemo-therapy associatedneuropathy. In some embodiments, enhanced bioavailability of thezoledronic acid may be achieved in treating one of these conditions byadministering a dosage form comprising zoledronic acid in the form of adisodium salt. This may allow a reduced molar amount of the disodiumsalt to be used as compared to what would be used with the diacid form.

In some embodiments, administration of a RANK/RANKL antagonist, such asan antibody (e.g., denosumab, a biological product that isinterchangeable with denosumab, a biosimilar of denosumab, or anantibody which competes with denosumab for binding to RANKL), or a smallmolecule such as ibrutinib, zoledronic acid, or minodronic acid, mayalso be useful to treat hypercalcemia of malignancy, multiple myeloma,bone metastases from solid tumors, Paget's disease of bone, giant celltumor of bone, blood cancers or leukemias, or solid tumors or cancers.

In some embodiments, the mammal being treated may not be suffering frombone metastasis. In some embodiments, the mammal being treated may notbe suffering from cancer. In some embodiments, the mammal being treatedmay not be suffering from osteoporosis. In some embodiments, if themammal has osteoporosis or cancer, the mammal is being treated for painthat is not a result of osteoporosis or cancer. In some embodiments, ifthe mammal has osteoporosis or cancer, a second therapeutic agent isadministered to the mammal for the treatment of osteoporosis or cancer,and wherein the second therapeutic agent is not a RANK/RANKL antagonist.

A RANK/RANKL antagonist, such as an antibody (e.g., denosumab, abiological product that is interchangeable with denosumab, a biosimilarof denosumab, or an antibody which competes with denosumab for bindingto RANKL), or a small molecule such as ibrutinib, zoledronic acid, orminodronic acid, may also be administered to relieve cancer-relatedpain, including pain associated with multiple myeloma and bonemetastases from solid tumors. In some embodiments, a RANK/RANKLantagonist is used to treat pain that may not be cancer-related pain.For example, a RANK/RANKL antagonist may be used to treat pain that maynot be associated with multiple myeloma, bone metastasis from solidtumors, hypercalcemia of malignancy, giant cell tumor of bone, bloodcancers or leukemias, or solid tumors or cancers. In some embodiments,enhanced bioavailability of zoledronic acid or minodronic acid may beachieved in treating one of these conditions by administering a dosageform comprising zoledronic acid or minodronic acid in the form of adisodium salt. This may allow a reduced molar amount of the disodiumsalt to be used as compared to what would be used with the diacid form.

In some embodiments, a RANK/RANKL antagonist, such as an antibody (e.g.,denosumab, a biological product that is interchangeable with denosumab,a biosimilar of denosumab, or an antibody which competes with denosumabfor binding to RANKL), or a small molecule such as ibrutinib, zoledronicacid, or minodronic acid, may be administered to relieve complexregional pain syndrome, such as complex regional pain syndrome type I(CRPS-I), complex regional pain syndrome type II (CRPS-II), CRPS-NOS, oranother type of CRPS. CRPS is a type of inflammatory pain. CRPS can alsohave a neuropathic component.

Complex regional pain syndrome is a debilitating pain syndrome. It ischaracterized by severe pain in a limb accompanied by edema, andautonomic, motor and sensory changes.

In some embodiments, a RANK/RANKL antagonist, such as an antibody (e.g.,denosumab, a biological product that is interchangeable with denosumab,a biosimilar of denosumab, or an antibody which competes with denosumabfor binding to RANKL), or a small molecule such as ibrutinib, zoledronicacid, or minodronic acid, may be administered to treat fibromyalgia.Fibromyalgia is a condition which may affect the muscles and softtissue, and may include symptoms in the head, back, neck, shoulder,and/or hip.

In some embodiments, a RANK/RANKL antagonist, such as an antibody (e.g.,denosumab, a biological product that is interchangeable with denosumab,a biosimilar of denosumab, or an antibody which competes with denosumabfor binding to RANKL), or a small molecule such as ibrutinib, zoledronicacid, or minodronic acid, may also be useful to treat complex regionalpain syndrome, rheumatoid arthritis, osteoarthritis, erosiveosteoarthritis, axial spondyloarthritis including ankylosingspondylitis, acute vertebral crush fracture, fibrous dysplasia, SAPHOsyndrome, osteoporosis, transient osteoporosis, or transientosteoporosis of the hip. In some embodiments, enhanced bioavailabilityof zoledronic acid or minodronic acid may be achieved in treating one ofthese conditions by administering a dosage form comprising zoledronicacid or minodronic acid in the form of a disodium salt. This may allow areduced molar amount of the disodium salt to be used as compared to whatwould be used with the diacid form.

In some embodiments, administration a RAN K/RANKL antagonist, such as anantibody (e.g., denosumab, a biological product that is interchangeablewith denosumab, a biosimilar of denosumab, or an antibody which competeswith denosumab for binding to RANKL) or an oral small molecule (such asibrutinib, zoledronic acid, or minodronic acid) may also be useful totreat hypercalcemia of malignancy, multiple myeloma, bone metastasesfrom solid tumors, Paget's disease of bone, giant cell tumor of bone,blood cancers or leukemias, or solid tumors or cancers. In someembodiments, enhanced bioavailability of zoledronic acid or minodronicacid may be achieved in treating one of these conditions byadministering a dosage form comprising zoledronic acid or minodronicacid in the form of a disodium salt. This may allow a reduced molaramount of the disodium salt to be used as compared to what would be usedwith the diacid form.

With respect to the treatment of any condition recited herein, in someembodiments a first dose of the RAN K/RANKL antagonist is administeredand a second dose of the RANK/RANKL antagonist is administered. Thetiming of the administration of the two doses may be such that, withrespect to the first dose, the second dose with respect to the firstdose, the second dose is administered at 5×T_(max) or greater (e.g., ifT_(max) is 1 hour, at 5 hours or later), at least 10×T_(max) or greater,at least about 15×T_(max) or greater, at least about 20×T_(max) orgreater, at least about 50×T_(max) or greater, or at least about200×T_(max) or greater, wherein T_(max) is the time of maximum plasmaconcentration for the first dose.

Some embodiments include treatment of a condition recited herein, suchas musculoskeletal pain, inflammatory pain, arthritis, or complexregional pain syndrome, wherein the treatment comprises either:administering only one dose or dosage form to a mammal to treat thecondition, or administering a first dose or dosage form to the mammal(e.g. orally), followed by administering a second dose or dosage form tothe mammal (e.g. orally). If two or more doses or dosage forms areadministered, the second dose or dosage form is administered before themaximum pain relieving effect of the first dose or dosage form isachieved, or before a peak in the pain relieving effect of the firstdose or dosage form is experienced by a mammal, receiving the dose ordosage form. In some embodiments, the second dose or dosage form isadministered before an observable pain relieving effect is achieved. Insome embodiments, the second dose or dosage form is administered about12 hours to about 60 days, about 24 hours to about 28 days, about 24hours to about 7 days, about 24 hours to about 14 days, or about 24hours to about 21 days, after the first dose or dosage form isadministered.

Some embodiments include treatment of a condition recited herein, suchas musculoskeletal pain, inflammatory pain, arthritis, or complexregional pain syndrome, wherein the treatment comprises administering afirst dose or dosage form, such as a first oral dosage form, to themammal, followed by administering a second dose or dosage form, such asa second oral dosage form, to the mammal, wherein the second dose ordosage form is administered after the maximum pain relieving effect ofthe first dose or dosage form is achieved, and the second dose or dosageform is administered while the mammal is still experiencing pain relieffrom the first dose or dosage form, or while the pain relieving effectfrom the first dose or dosage form is observable. In some embodiments,the second dose or dosage form is administered about 12 hours to about60 days, about 24 hours to about 28 days, about 24 hours to about 7days, about 24 hours to about 14 days, or about 24 hours to about 21days, after the first dose or dosage form is administered.

In addition to relieving pain, administration of a RANK/RANKLantagonist, such as an antibody (e.g., denosumab, a biological productthat is interchangeable with denosumab, a biosimilar of denosumab, or anantibody which competes with denosumab for binding to RANKL), or a smallmolecule such as ibrutinib, zoledronic acid, or minodronic acid, mayalso be useful to treat diseases or conditions that may or may notinclude a pain component. For example, a RANK/RANKL antagonist may beuseful to treat any of the pain conditions or types of conditions listedabove, including treatment that does not simply relieve the pain ofthose conditions, and treatment that is carried out in such a way thatthe condition is treated without pain relief occurring. In addition toany pain relief a RANK/RANKL antagonist may or may not provide, aRANK/RANKL antagonist may be used to treat a disease or condition suchas a metabolic disease or condition; an inflammatory disease orcondition, including an inflammatory disease or condition that might notbe associated with pain; a cancer disease or condition; a neurologicaldisease or condition; etc.

The duration of treatment will vary depending upon the particularRANK/RANKL antagonist, the disease or condition being treated, and otherfactors. For biological molecules such as denosumab, a biologicalproduct that is interchangeable with denosumab, a biosimilar ofdenosumab, or an antibody which competes with denosumab for binding toRANKL, repeated doses may be administered over at least a period of twoweeks or longer, or may be administered indefinitely. Several rounds oftreatment may be given, alternating with periods of no treatment. Ifdiscontinued, treatment may be resumed if a relapse of the pain shouldoccur.

For therapeutic use, a RANK/RANKL antagonist can be administered to amammal, including a human patient, for treatment in a manner appropriateto the indication. Systemic administration may be used. The RANK/RANKLantagonist may be applied locally. For biological molecules such asdenosumab, a biological product that is interchangeable with denosumab,a biosimilar of denosumab, or an antibody which competes with denosumabfor binding to RANKL, means of local administration include, forexample, local injection, or application of the antagonist admixed orpolymerized with a slow-release matrix suitable for this purpose, manyof which can be known.

In some embodiments, RANK/RANKL antagonists can be concurrentlyadministered with other drugs or therapeutic agents in the manufactureof a medicament for the treatment of pain. RANK/RANKL antagonists andother drugs may be formulated into therapeutic compositions comprisingan effective amount of the antagonist.

In some embodiments, therapeutic agents that may be administered inconjunction with the RANK/RANKL antagonists described herein may includetherapeutic agents indicated for the treatment of pain or anotherneurological disorder. In some embodiments, therapeutic agents indicatedfor the treatment of pain and/or inflammation may include analgesicand/or anti-inflammatory agents, including aspirin (acetylsalicylicacid), nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen,naproxen, ketoprofen, celecoxib, firocoxib, meloxicam, etc.),acetaminophen, narcotic or opioid pain relievers (e.g., morphine,codeine, fentanyl, oxycodone, hydrocodone, hydromorphone, etc.), andsteroids (e.g., triamcinolone, prednisone, methylprednisolone,cortisone, etc.).

In some embodiments, sustained-release forms of RANK/RANKL antagonistscan be used. Sustained-release forms suitable for use in the disclosedmethods include, but are not limited to, soluble RANK polypeptides, andantagonistic anti-RANK or anti-RANKL antibodies that can be encapsulatedin a slowly-dissolving biocompatible polymer (such as the alginatemicroparticles described in U.S. Pat. No. 6,036,978), admixed with aslow-release polymer (including topically applied hydrogels), and/orincorporated into a biocompatible semi-permeable implant.

The amount of RANK/RANKL antagonist administered per dose will varydepending on the antagonist being used and the mode of administration.If the antagonist is a soluble RANK and is administered by injection,the effective amount per adult dose will range from about 0.5 mg/m² toabout 20 mg/m², about 1 mg/m² to about 5 mg/m², about 3 mg/m² to about10 mg/m², about 5 mg/m² to about 10 mg/m², about 7 mg/m² to about 12mg/m², about 10 mg/m² to about 20 mg/m², about 15 mg/m² to about 25mg/m², about 0.5 mg/m² to about 10 mg/m², or from about 5 mg/m² to about12 mg/m² based on the body surface area of the mammal. Alternatively, aflat dose may be administered, whose amount may range from 5 mg/dose toabout 100 mg/dose, about 5 mg/dose to 50 mg/dose, about 10 mg/dose to 60mg/dose, about 15 mg/dose to about 45 mg/dose, about 50 mg/dose to about100 mg/dose, about 20 mg/dose to about 70 mg/dose, about 30 mg/dose toabout 75 mg/dose, about 25 mg/dose to about 50 mg/dose, or any dose inbetween or bounded by these ranges. Some dose ranges for a flat dose tobe administered by subcutaneous injection can be 5 mg/dose to about 25mg/dose, 25 mg/dose to about 50 mg/dose, about 15 mg/dose to about 30mg/dose, about 45 mg/dose to about 70 mg/dose, about 60 mg/dose to about80 mg/dose, about 1 mg/dose to about 20 mg/dose, about 30 mg/dose toabout 60 mg/dose, or about 50 mg/dose to about 100 mg/dose. The chosendose may be administered repeatedly, particularly for chronicconditions, or the amount per dose may be increased or decreased astreatment progresses. The chosen dose may be administered one or moretimes per week, monthly, every two months, every three months, every sixmonths, or every year.

For pediatric patients (ages 4-17), a suitable regimen involves thesubcutaneous injection of 0.4 mg/kg, up to a maximum dose of 25 mg ormore, about 0.1 mg/kg to about 10 mg/kg, about 5 mg/kg to about 15mg/kg, about 10 mg/kg to about 25 mg/kg, about 10 mg/kg to about 17mg/kg, about 12 mg/kg to about 20 mg/kg, about 15 mg/kg to about 25mg/kg, or about 15 mg/kg to about 40 mg/kg, to be administered one ormore times per week, monthly, every two months, every three months,every six months, or every year. If an antibody against RANK or RANKL isused as the RANK/RANKL antagonist, useful dose ranges include about 0.1mg/kg to about 20 mg/kg, about 0.75 mg/kg to about 7.5 mg/kg and about 1mg/kg to about 10 mg/kg of body weight. The chosen dose may beadministered repeatedly, particularly for chronic conditions, or theamount per dose may be increased or decreased as treatment progresses.The chosen dose may be administered one or more times per week, monthly,every two months, every three months, every six months, or every year.

Useful doses for an antibody against RANK or RANKL, such as denosumab,may range from about 0.1 mg/kg to about 20 mg/kg, about 0.75 mg/kg toabout 7.5 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 1 mg/kg toabout 2 mg/kg, about 10 mg/kg to about 20 mg/kg, about 12 to about 17mg/kg, about 15 mg/kg to about 20 mg/kg, about 1 mg/kg, about 1 mg/kg toabout 10 mg/kg, or any value bounded by or in between these ranges basedon the body weight of the mammal. The chosen dose may be administeredrepeatedly, particularly for chronic conditions, or the amount per dosemay be increased or decreased as treatment progresses. The chosen dosemay be administered one or more times per week, monthly, every twomonths, every three months, every six months, or every year.

Doses of the antagonist may be administered daily, weekly, monthly,every three months, every six months, or every year. Doses may beadministered in single or divided doses. In some embodiments, humanizedantibodies are used, that is, antibodies in which only theantigen-binding portion of the antibody molecule is derived from anon-human source. Antibodies may be administered by injection, includingintravenous infusion. Appropriate dosages can be determined in trials.The amount and frequency of administration will depend, of course, onsuch factors as the nature and severity of the indication being treated,the desired response, the condition of the patient, and so forth.

Ibrutinub may be administered orally in a therapeutically effectiveamount, such as about 1 mg to about 1000 mg, about 1 mg to about 500 mg,about 1 mg to 100 mg, about 1 mg to about 10 mg, 20 mg to about 30 mg,30 mg to about 40 mg, 40 mg to about 50 mg, 50 mg to about 60 mg, 60 mgto about 70 mg, 70 mg to about 80 mg, 90 mg to about 100 mg, about 100mg to 1000 mg, about 200 mg to about 700 mg, about 400 mg to about 600mg, about 560 mg, or about 420 mg. These amounts may be administereddaily, weekly, monthly, etc. In some embodiments, ibrutinub may beadministered once daily.

In some embodiments the daily dose, such as the daily oral dose, ofzoledronic acid or minodronic acid is about 0.005 mg to about 20 mg,about 0.1 mg to about 10 mg, about 0.5 mg to about 10 mg, about 0.2 mgto about 5 mg, about 0.2 mg to about 3 mg, about 0.5 mg to about 5 mg,about 0.5 mg to about 3 mg, about 0.5 mg to about 3 mg, or any amount ofzoledronic acid or minodronic acid in a range bounded by, or between,any of these values. In some embodiments, the daily dose, such as thedaily oral dose, of zoledronic acid or minodronic acid is less thanabout 35 mg/m², less than about 30 mg/m², less than about 25 mg/m²,about 1 mg/m² to about 35 mg/m², about 1 mg/m² to about 30 mg/m², about1.5 mg/m² to about 25 mg/m², about 1.8 mg/m² to about 20 mg/m², about 10mg/m² to about 20 mg/m², about 10 mg/m² to about 30 mg/m², about 15mg/m² to about 20 mg/m², about 18 mg/m², or any amount of zoledronicacid or minodronic acid in a range bounded by, or between, any of thesevalues.

In some embodiments the weekly dose, such as the weekly oral dose, ofzoledronic acid or minodronic acid can be about 1 mg to about 1000 mg,about 1 mg to about 500 mg, about 10 mg to about 250 mg, about 100 mg toabout 300 mg, about 10 mg to about 100 mg, about 10 mg to about 80 mg,about 40 mg to about 60 mg, about 10 mg to about 150 mg, about 10 mg toabout 100 mg, about 10 mg to about 300 mg, about 20 mg to about 150 mg,or about 30 mg to about 100 mg. In some embodiments, the weekly dose,such as the weekly oral dose, of zoledronic acid or minodronic acid canbe less than about 250 mg/m², less than about 200 mg/m², less than about175 mg/m², about 6 mg/m² to about 250 mg/m², about 10 mg/m² to about 210mg/m², about 10 mg/m² to about 170 mg/m², about 4 mg/m² to about 140mg/m², about 10 mg/m² to about 100 mg/m², about 50 mg/m² to about 100mg/m², about 70 mg/m² to about 90 mg/m², about 100 mg/m² to about 140mg/m², about 126 mg/m², or any amount of zoledronic acid or minodronicacid in a range bounded by, or between, any of these values. The weeklyoral dose may be given as a single dose, given once during the week, ormay be given in 2, 3, 4, 5, 6, or 7 individual doses during the week. Insome embodiments, a weekly dose is given for 4, 5, 6, 7, 8, 9, 10, 11,or 12 consecutive weeks.

In some embodiments, the monthly dose, such as the monthly oral dose, ofzoledronic acid or minodronic acid, or the amount of zoledronic acid orminodronic acid that can be administered over a period of a month, isabout 5000 mg or less, about 4000 mg or less, about 3000 mg or less,about 2000 mg or less, about 1000 mg or less, about 700 mg or less,about 600 mg or less, about 1 mg to about 4,000 mg, about 1 mg to about1,000 mg, about 10 mg to about 1000 mg, about 50 mg to about 1000 mg,about 10 mg to about 600 mg, about 10 mg to about 300 mg, about 100 mgto about 300 mg, about 100 mg to about 200 mg, about 200 mg to about 300mg, about 40 mg to about 600 mg, about 50 mg to about 600 mg, about 200mg to about 400 mg, or about 100 mg to about 600 mg, about 40 mg toabout 2000 mg, about 40 mg to about 800 mg, about 50 mg to about 800 mg,or about 100 mg to about 800 mg, about 40 mg to about 1000 mg, about 50mg to about 1000 mg, or about 100 mg to about 1000 mg, or any monthlydose in a range bounded by, or between, any of these values.

In some embodiments, the monthly dose, such as the monthly oral dose, ofzoledronic acid or minodronic acid can be less than about 1000 mg/m²,less than about 800 mg/m², less than about 600 mg/m², about 10 mg/m² toabout 1000 mg/m², about 50 mg/m² to about 800 mg/m², about 70 mg/m² toabout 700 mg/m², about 100 mg/m² to about 700 mg/m², about 100 mg/m² toabout 600 mg/m², about 50 mg/m² to about 500 mg/m², about 200 mg/m² toabout 500 mg/m², about 100 mg/m² to about 400 mg/m², about 200 mg/m² toabout 400 mg/m², about 50 mg/m² to about 200 mg/m², about 300 mg/m² toabout 600 mg/m², about 450 mg/m² to about 600 mg/m², about 300 mg/m² toabout 1000 mg/m², about 400 mg/m² to about 1000 mg/m², about 500 mg/m²to about 1000 mg/m², about 400 mg/m² to about 700 mg/m², about 500 mg/m²to about 600 mg/m², about 540 mg/m², or any amount of zoledronic acid orminodronic acid in a range bounded by, or between, any of these values.

Minodronic acid may be administered, e.g. intravenously, in atherapeutically effective amount, such as 0.1 mg to about 5 mg, about0.5 mg to about 2 mg, or about 1 mg, about 1 mg to about 10 mg, about 2mg to about 6 mg, or about 4 mg, about 0.5 mg to about 60 mg, about 4 mgto about 60 mg, or about 40 mg to about 60 mg. The chosen dose may beadministered repeatedly, particularly for chronic conditions, or theamount per dose may be increased or decreased as treatment progresses.The chosen dose may be administered one or more times per week, monthly,every two months, every three months, every six months, or every year.In some embodiments, minodronic acid may be administered in an amountthat is about 0.1 mg to about 5 mg, about 0.5 mg to about 2 mg, or about1 mg per week; about 1 mg to about 10 mg, about 2 mg to about 6 mg, orabout 4 mg per month; about 0.5 mg to about 60 mg, about 4 mg to about60 mg, or about 40 mg to about 60 mg per year.

A monthly dose, such as the monthly oral dose, may be given as a singledose, or as two or more individual doses administered during the month.In some embodiments, the monthly dose, such as the monthly oral dose,can be administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 individual dosesduring the month. In some embodiments, the monthly dose, such as themonthly oral dose, can be administered in 2 or 3 weekly doses. In someembodiments, the monthly dose, such as the monthly oral dose, can beadministered in 4 or 5 weekly doses. In some embodiments, the monthlydose, such as the monthly oral dose, can be administered in 28 to 31daily doses. In some embodiments, the monthly dose, such as the monthlyoral dose, can be administered in 5 to 10 individual doses during themonth. The monthly dose, such as the monthly oral dose, may beadministered for only 1 month, or may be repeatedly administered for 2,3, 4, 5, 6 or more months.

The RANK/RANKL antagonists may be administered orally, parenterally,sublingually, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. Topicaladministration may also involve the use of transdermal administrationsuch as transdermal patches or ionophoresis devices. Injection can be aroute of administration that may be used, including parenteralinjection. Parenteral injections include subcutaneous injections,intraspinal, intrathecal, intraorbital, intravenous, intrarterial,intramuscular, intrasternal, and infusion techniques. Compositionscomprising a RANK/RANKL antagonist can be administered by bolusinjection or continuous infusion. Routes of systemic administration thatmay be used include subcutaneous injection and intravenous drip.

Pharmaceutical compositions suitable for parenteral administration(e.g., by injection, for example bolus injection or continuous infusion)and may be presented in unit dose form in ampoules, pre-filled syringes,small volume infusion or in multi-dose containers with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution, for constitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use.

With respect to oral administration of zoledronic acid or minodronicacid for the treatment of pain associated with inflammation, arthritis,CRPS, or any other condition recited herein, it may helpful if themammal or human being to which the zoledronic acid is administered doesnot eat food or drink beverage, (other than any water required toswallow the oral dosage form) for at least about 1 hour, at least about2 hours, at least about 4 hours, at least about 6 hours, at least about8 hours, at least about 10 hours, or at least about 12 hours before thezoledronic acid is administered. It may also be helpful if the mammal orhuman being to which the zoledronic acid is administered does not eatfood or drink beverage for at least about 30 minutes, at least about 1hour, at least about 2 hours, at least about 3 hours, or at least about4 hours after the zoledronic acid is administered. In some embodiments,a human being to which the zoledronic acid is administered avoids lyingdown, or remains upright or sits upright, for at least about 30 minutesor about 1 hour after receiving a dosage form containing zoledronicacid. Avoiding food or beverage before or after oral administration ofzoledronic acid can improve the bioavailability of the zoledronic acid.

The effective amount of zoledronic acid or minodronic acid will varydepending on various factors known to the treating physicians, such asthe severity of the condition to be treated, route of administration,formulation and dosage forms, and age, weight and response of theindividual patients.

RANK/RANKL antagonists and other drugs may be formulated intotherapeutic compositions comprising an effective amount of theantagonist.

Some embodiments include a pharmaceutical composition comprising apurified soluble protein having RANK/RANKL antagonistic activity, inconjunction with physiologically acceptable carriers, excipients ordiluents. Such carriers may be nontoxic to recipients at the dosages andconcentrations employed. Inhibitors of the RANK/RANKL interaction forpharmaceutical compositions can be complexed with polyethylene glycol(PEG), metal ions, or incorporated into polymeric compounds such aspolyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts.

Protein complexes with PEG can be made using known procedures, such asfor example, those described in U.S. Pat. Nos. 5,849,860, 5,766,897 orother suitable methods. Suitable lipids for liposomal formulationinclude, without limitation, monoglycerides, diglycerides, cholesterol,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; 4,737,323; and 5,858,397. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance, and can be thus chosen accordingto the intended application, so that the characteristics of the carrierwill depend on the selected route of administration.

Ordinarily, the preparation of pharmaceutical compositions comprising aRANK/RANKL antagonist that is a protein entails combining thetherapeutic protein with buffers, antioxidants such as ascorbic acid,low molecular weight (less than about 10 residues) polypeptides,proteins, amino acids, carbohydrates including glucose, sucrose ordextrins, chelating agents such as EDTA, glutathione and otherstabilizers and excipients. Neutral buffered saline or saline mixed withconspecific serum albumin can be exemplary appropriate diluents. Incertain embodiments, the product can be formulated as a lyophilizateusing appropriate excipient solutions (e.g., sterile water or sucrosesolution) as diluents. One embodiment entails packaging a lyophilizedRANK/RANKL antagonist in dose unit form which when reconstituted willprovide one to three doses per package.

In one aspect at least one RANK/RANKL antagonist or a pharmaceuticallyacceptable salt thereof or composition comprising same can be used incombination with another therapy indicated for pain or otherneurological disorders.

Small molecule RANK/RANKL antagonists, including ibrutinib, zoledronicacid, and minodronic acid, may be formulated for oral administration,for example, with an inert diluent or with an edible carrier, or it maybe enclosed in hard or soft shell gelatin capsules, compressed intotablets, or incorporated directly with the food of the diet. For oraltherapeutic administration, the active compound may be incorporated withan excipient and used in the form of ingestible tablets, buccal tablets,coated tablets, troches, capsules, elixirs, dispersions, suspensions,solutions, syrups, wafers, patches, and the like.

Tablets, troches, pills, capsules and the like may also contain one ormore of the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; an excipient, such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, lactose or saccharin; or a flavoring agent such aspeppermint, oil of wintergreen or cherry flavoring. When the unit dosageform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier. Various other materials may be present ascoating, for instance, tablets, pills, or capsules may be coated withshellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Itmay be desirable for material in a dosage form or pharmaceuticalcomposition to be pharmaceutically pure and substantially non toxic inthe amounts employed.

Some compositions or dosage forms may be a liquid, or may comprise asolid phase dispersed in a liquid.

Zoledronic acid may be formulated for parenteral or intraperitonealadministration. Solutions of the active compounds as free acids orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. A dispersioncan also have an oil dispersed within, or dispersed in, glycerol, liquidpolyethylene glycols, and mixtures thereof. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

In some embodiments, sustained-release forms of RANK/RANKL antagonistscan be used. Sustained-release forms suitable for use in the disclosedmethods include, but are not limited to, soluble RANK polypeptides, andantagonistic anti-RANK or anti-RANKL antibodies that can be encapsulatedin a slowly-dissolving biocompatible polymer (such as the alginatemicroparticles described in U.S. Pat. No. 6,036,978), admixed with aslow-release polymer (including topically applied hydrogels), and/orincorporated into a biocompatible semi-permeable implant.

The amount of the RANK/RANKL antagonist, such as denosumab, or a smallmolecule such as ibrutinib, zoledronic acid, or minodronic acid, in atherapeutic composition may vary. For example, some liquid compositionsmay comprise about 0.0001% (w/v) to about 50% (w/v), about 0.01% (w/v)to about 20% (w/v), about 0.01% to about 10% (w/v), about 0.001% (w/v)to about 1% (w/v), about 0.1% (w/v) to about 0.5% (w/v), about 1% (w/v)to about 3% (w/v), about 3% (w/v) to about 5% (w/v), about 5% (w/v) toabout 7% (w/v), about 7% (w/v) to about 10% (w/v), about 10% (w/v) toabout 15% (w/v), about 15% (w/v) to about 20% (w/v), about 20% (w/v) toabout 30% (w/v), about 30% (w/v) to about 40% (w/v), or about 40% (w/v)to about 50% (w/v) of the RANK/RANKL antagonist.

Some solid compositions may comprise at least about 5% (w/w), at leastabout 10% (w/w), at least about 20% (w/w), at least about 50% (w/w), atleast about 70% (w/w), at least about 80%, about 10% (w/w) to about 30%(w/w), about 10% (w/w) to about 20% (w/w), about 20% (w/w) to about 30%(w/w), about 30% (w/w) to about 50% (w/w), about 30% (w/w) to about 40%(w/w), about 40% (w/w) to about 50% (w/w), about 50% (w/w) to about 80%(w/w), about 50% (w/w) to about 60% (w/w), about 70% (w/w) to about 75%(w/w), about 70% (w/w) to about 80% (w/w), or about 80% (w/w) to about90% (w/w) of a RANK/RANKL antagonist such as a ibrutinib, zoledronicacid, or minodronic acid.

Any suitable amount of RANK/RANKL antagonist, such as denosumab,ibrutinib, zoledronic acid, or minodronic acid, may be used. Some solidor liquid oral dosage forms, or units of oral dosage forms (referred tocollectively herein as “oral dosage form(s)”) may contain about 0.005 mgto about 20 mg, about 0.1 mg to about 10 mg, about 0.5 mg to about 10mg, about 0.2 mg to about 5 mg, about 1 mg to about 500 mg, about 1 mgto about 50 mg, about 1 mg to about 75 mg, about 10 mg to about 250 mg,about 100 mg to about 300 mg, about 20 mg to about 200 mg, about 20 mgto about 150 mg, about 30 mg to about 100 mg, about 30 mg to about 150mg, about 1 mg to about 1,000 mg, about 10 mg to about 50 mg, about 10mg to about 300 mg, about 10 mg to about 150 mg, about 10 mg to about100 mg, about 40 mg to about 150 mg, about 40 mg to about 220 mg, about10 mg to about 600 mg, about 40 mg to about 600 mg, about 40 mg to about2000 mg, about 40 mg to about 800 mg, about 25 mg to about 800 mg, about30 mg to about 800 mg, about 10 mg to about 500 mg, about 50 mg to about150 mg, about 50 mg, about 100 mg, about 50 mg to about 500 mg, about100 mg to about 2000 mg, about 300 mg to about 1500 mg, about 200 mg toabout 1000 mg, about 100 mg to about 500 mg, or about 150 mg ofzoledronic acid or minodronic acid, or any amount of RANK/RANKLantagonist in a range bounded by, or between, any of these values. Insome embodiments, the RANK/RANKL antagonist can be administered daily,weekly, monthly, every two or three months, once a year, or twice ayear.

In some embodiments, an oral dosage form may contain about 10 mg/m² toabout 20 mg/m², about 15 mg/m² to about 20 mg/m², about 18 mg/m², about80 mg/m² to about 150 mg/m², about 90 mg/m² to about 150 mg/m², about100 mg/m² to about 150 mg/m² of zoledronic acid or minodronic acid, orany amount of the compound in a range bounded by, or between, any ofthese values. All dosage ranges or amounts expressed in mg/m² can bebased upon the body surface area of the mammal.

Oral zoledronic acid, or disodium salt thereof, may be administered incombination with about 0.1 mg to about 10 mg of zoledronic acid, or asalt thereof, administered parenterally, such as intravenously. In someembodiments, about 50 mg, about 100 mg, or about 150 mg of the disodiumsalt of zoledronic acid can be administered orally in combination with 1mg parenteral, such as intravenous, zoledronic acid. In some embodimentsthe parenteral dose of zoledronic acid can be about 0.25 mg to about 25mg, about 0.25 mg to about 10 mg, or about 0.5 mg to about 7.5 mg.

The oral bioavailability of zoledronic acid or minodronic acid may beenhanced by orally administering the zoledronic acid or minodronic acidin the disodium salt form.

For example, the bioavailability of zoledronic acid may be improved byat least about 10%, at least about 20%, at least about 30%, at leastabout 50%, and/or up to about 100%, or up to about 200%, as compared toadministration of zoledronic acid in the diacid form.

Similarly, the bioavailability of minodronic acid may be improved by atleast about 10%, at least about 20%, at least about 30%, at least about50%, and/or up to about 100%, or up to about 200%, as compared toadministration of minodronic acid in the diacid form.

Because of the improved bioavailability of the disodium salt a dosageform may contain, or a mammal, such as a human being, may receive, on amolar basis, less of the disodium salt form of zoledronic acid orminodronic acid than would otherwise be administered of the diacid formof zoledronic acid.

For example, a dosage form may contain, or a mammal may receive, atleast about 10 mole % less, at least about 20 mole % less, at leastabout 40 mole % less, at least about 50 mole % less, and/or up to about90 mole % less or 95 mole % less, of the disodium salt form as comparedthe amount of the diacid form of zoledronic acid that would otherwise beadministered, such as a molar amount that would be administered ofzoledronic acid in the diacid form in order to achieve the same plasmalevels of zoledronic acid.

Similarly, a dosage form may contain, or a mammal may receive, at leastabout 10 mole % less, at least about 20 mole % less, at least about 40mole % less, at least about 50 mole % less, and/or up to about 90 mole %less or 95 mole % less, of the disodium salt form as compared the amountof the diacid form of minodronic acid that would otherwise beadministered, such as a molar amount that would be administered ofminodronic acid in the diacid form in order to achieve the same plasmalevels of minodronic acid.

In some embodiments, a dosage form contains, or a mammal (such as ahuman being) can be administered, an amount of the disodium salt form,on a molar basis, that has a value of about 0.8 n_(d) to about 1.2 n_(d)or about 0.9 n_(d) to about 1.1 n_(d), wherein:n _(d)=(b _(a) /b _(d))(n _(a))wherein b_(a) is the bioavailability of the diacid form, b_(d) is thebioavailability of the disodium salt form, and n_(a) is the number ofmoles of the diacid that would be administered in a dosage formcontaining the diacid form of zoledronic acid. For example, if thediacid form has a bioavailability (b_(a)) of 0.01 and the disodium saltform has a bioavailability (b_(d)) of 0.015, and a dosage form wouldnormally contain 0.001 moles of the diacid, n_(d) would be(0.01/0.015)(0.001 moles), or about 0.00067 moles. In some embodiments,the disodium salt can be administered in an amount that has a value ofabout n_(d).

With respect to oral dosage forms comprising a reduced molar amount ofthe disodium salt of zoledronic acid as compared to the diacid form ofzoledronic acid, in some embodiments, the bioavailability of thezoledronic acid in the disodium salt form can be sufficiently high that,if the drug is administered to a mammal, at least as much zoledronicacid is present in the blood of the mammal as would be present ifzoledronic acid were administered in the diacid form.

With respect to oral dosage forms comprising the disodium salt form ofzoledronic acid, in some embodiments, the disodium salt form can bepresent in a lower molar amount than would be present if the zoledronicacid were in the diacid form; and the zoledronic acid in the disodiumsalt form has an improved bioavailability as compared to the zoledronicacid in the diacid form to the extent that the lower molar amount of thedisodium salt in the dosage form does not reduce the amount ofzoledronic acid delivered to the plasma of a mammal.

In some embodiments, the zoledronic acid in the disodium salt form canbe present in an amount such that the oral dosage form provides an areaunder the plasma concentration curve of zoledronic acid of about 4ng·h/mL to about 2000 ng·h/mL to the mammal each time the zoledronicacid in the disodium salt is administered.

In some embodiments, the zoledronic acid in the disodium salt form ispresent in an amount such that the oral dosage form provides an areaunder the plasma concentration curve of zoledronic acid of about 100ng·h/mL to about 2000 ng·h/mL, about 100 ng·h/mL to about 1000 ng·h/mL,about 500 ng·h/mL to about 1000 ng·h/mL, or about 500 ng·h/mL to about700 ng·h/mL in the mammal to which the dosage form is administered. Thisamount may be suitable for administration of the oral dosage form aboutevery 3 to 4 weeks.

In some embodiments, the zoledronic acid in the disodium salt form canbe present in an amount such that the oral dosage form provides an areaunder the plasma concentration curve of zoledronic acid of about 20ng·h/mL to about 700 ng·h/mL, about 50 ng·h/mL to about 500 ng·h/mL, orabout 100 ng·h/mL to about 200 ng·h/mL, in the mammal to which thedosage form is administered. This amount may be suitable for weeklyadministration of the oral dosage, or for administration of 3 to 5individual dosages during a month. The individual dosages could be givenat regular intervals, given during the first week, or at any otherschedule that provides 3 to 5 dosages during the month.

In some embodiments, the zoledronic acid in the disodium salt form canbe present in an amount such that the oral dosage form provides an areaunder the plasma concentration curve of zoledronic acid of about 4ng·h/mL to about 100 ng·h/mL, about 10 ng·h/mL to about 50 ng·h/mL, orabout 10 ng·h/mL to about 30 ng·h/mL, in the mammal to which the dosageform is administered. This amount may be suitable for dailyadministration of the oral dosage form.

Oral administration of zoledronic acid, particularly oral administrationof the disodium salt form of zoledronic acid, can result in moresustained plasma levels of the drug as compared to parenteral modes ofadministration, such intravenous or subcutaneous. For example, theamount of zoledronic acid in the plasma can be significantly higher fororal administration of the disodium salt about 24 hours or 48 hours, orlonger, after administration. In some embodiments, oral zoledronic acidhas a 24 hour sustained plasma level factor of about 1 or higher, suchas about 1 to about 10, about 1 to about 5, about 3 to about 5, or about3 to about 4. In some embodiments, an orally administered dosage form ofzoledronic acid has a 24 hour sustained plasma level factor or a 48 hoursustained plasma level factor that can be higher, such as at least 1.2times, at least about 2 times, at least about 5 times, about 1.2 timesto about 20 times, about 2 times to about 15 times, about 5 times toabout 10 times, or about 8 to about 15 times that of intravenouslyadministered zoledronic acid. A “sustained plasma level factor”, p_(f),can be determined by the equation:p _(f)=1000(C _(t) /C _(max))wherein C_(max) is the maximum plasma concentration of zoledronic acidafter it is administered and C_(t) is the plasma concentration ofzoledronic acid at the time of interest, such as 24 hours. Forparenteral administration, the C_(max) can be about the C₀, or theconcentration right after injection of the entire amount of the druginto the body. Sustained plasma level factors can also be obtained forother times, such as 48 hours, by using the plasma concentration ofzoledronic acid for C_(t) in the equation above. For example, if themaximum plasma level of zoledronic acid after administration is 1000ng/mL and the plasma level of zoledronic acid at 24 hours is 1 ng/mL,the 24 hour sustained plasma level factor is 1.

In some embodiments, the disodium salt form of zoledronic acid providesan enhancement to bioavailability, as compared to the diacid form ofzoledronic acid, which adds to any enhancement to bioavailabilityprovided by any bioavailability-enhancing agents in the dosage form. Insome embodiments, the disodium salt form of zoledronic acid provides anenhancement to bioavailability, as compared to the diacid form ofzoledronic acid, which is greater than any enhancement tobioavailability provided by any bioavailability-enhancing agents in thedosage form. In some embodiments, the disodium salt form of zoledronicacid may be administered in a dosage form that is substantially free ofbioavailability-enhancing agents.

In some embodiments, a dosage form comprising a disodium salt ofzoledronic acid is a solid.

In some embodiments, a dosage form comprising a disodium salt ofzoledronic acid can be used to treat an inflammatory condition.

In some embodiments, a dosage form comprising a disodium salt ofzoledronic acid can be used to treat arthritis.

In some embodiments, a dosage form comprising a disodium salt ofzoledronic acid can be used to treat complex regional pain syndrome.

In some embodiments, zoledronic acid can be in a form that has anaqueous solubility, meaning the solubility in water, greater than 1%(w/v), about 5% (w/v) to about 50% (w/v), about 5% (w/v) to about 20%(w/v), about 10% (w/v) to about 15% (w/v), or about 12% (w/v) to about13% (w/v).

The disodium salt form of zoledronic acid can be more compressible thanthe diacid form of zoledronic acid. This can make it easier for a dosageform to have a desired hardness. It can also make it easier to increasethe drug load, so that a smaller tablet can be given for a given dosagestrength. In some embodiments, a solid dosage form of zoledronic acid,such as the diacid form of zoledronic acid or the disodium salt form ofzoledronic acid, can have a hardness of about 5 kPa to about 20 kPa orabout 5 kPa to about 14 kPa.

The oral bioavailability of zoledronic acid in a dosage form can vary.Some dosage forms may have ingredients added to enhance thebioavailability. However, bioavailability enhancement might not benecessary for an oral dosage form to be effective. In some embodiments,the dosage form can be substantially free of bioavailability-enhancingagents. In some embodiments, an oral dosage form may have an oralbioavailability of zoledronic acid of about 0.01% to about 10%, about0.1% to about 7%, about 0.1% to about 5%, etc. Without ingredients orother methods to enhance bioavailability, zoledronic acid typically hasa low bioavailability in an oral dosage form. In some embodiments, theoral bioavailability of zoledronic acid can be unenhanced orsubstantially unenhanced. For example, the oral bioavailability ofzoledronic acid can be about 0.01% to about 5%, about 0.01% to about 4%,about 0.1% to about 3%, about 0.1% to about 2%, about 0.2% to about 2%,about 0.2% to about 1.5%, about 0.3% to about 1.5%, about 0.3% to about1%, about 0.1% to about 0.5%, about 0.3% to about 0.5%, about 0.5% toabout 1%, about 0.6% to about 0.7%, about 0.7% to about 0.8%, about 0.8%to about 0.9%, about 0.9%, about 1% to about 1.1%, about 1.1% to about1.2%, about 1.2% to about 1.3%, about 1.3% to about 1.4%, about 1.4% toabout 1.5%, about 1.5% to about 1.6%, about 1.6% to about 1.8%, or about1.8% to about 2%.

One embodiment is a pharmaceutical composition comprising zoledronicacid wherein the oral bioavailability of zoledronic acid in the dosageform is from about 0.01% to about 10%, about 0.01% to about 5%, about0.1% to about 7%, about 0.1% to about 5%, about 0.1% to about 3%, about0.1% to about 2%, about 0.2% to about 2%, about 0.2% to about 1.5%,about 0.3% to about 1.5%, about 0.3% to about 1.0%, about 1% to about2.5%, about 1.2% to about 2.5%, about 1.5% to about 2.5%, about 1% toabout 2.7%, about 1.2% to about 2.7%, about 1.5% to about 2.7%, about 1%to about 3%, about 1.2% to about 3%, about 1.5% to about 3%, about 1% toabout 3.5%, about 1.2% to about 3.5%, about 1.5% to about 3.5%,.

An oral dosage form comprising zoledronic acid or minodronic acid may beincluded in a pharmaceutical product comprising more than one unit ofthe oral dosage form.

In some embodiments, an oral dosage form comprises about 10 mg to about300 mg of zoledronic acid, and can be administered daily for about 2 toabout 15 consecutive days. This regimen may be repeated once monthly,once every two months, once every three months, once every four months,once every five months, once every six months, once yearly, or onceevery two years.

In some embodiments, an oral dosage form comprises about 10 mg to about150 mg or about 10 mg to about 100 mg of zoledronic acid, and can beadministered daily for about 2 to about 15 consecutive days. Thisregimen may be repeated once monthly, once every two months, once everythree months, once every four months, once every five months, once everysix months, once yearly, or once every two years.

In some embodiments, an oral dosage form comprises about 10 mg to about150 mg or about 10 mg to about 100 mg of zoledronic acid, and can beadministered daily for about 5 to about 10 consecutive days. Thisregimen may be repeated once monthly, once every two months, once everythree months, once every four months, once every five months, once everysix months, once yearly, or once every two years.

In some embodiments, an oral dosage form comprises about 40 mg to about150 mg of zoledronic acid, and can be administered daily for about 5 toabout 10 consecutive days. This regimen may be repeated once monthly,once every two months, once every three months, once every four months,once every five months, once every six months, once yearly, or onceevery two years.

In some embodiments, the oral zoledronic acid may be administered as onedose of about 100 mg to about 2000 mg. In some embodiments, the oralzoledronic acid may be administered as one dose of about 300 mg to about1500 mg. In some embodiments, the oral zoledronic acid may beadministered as one dose of about 200 mg to about 1000 mg. The dose ofzoledronic acid may be administered in a single or divided dose.

A pharmaceutical product containing oral dosage forms for daily use cancontain 28, 29, 30, or 31 units of the oral dosage form for a monthlysupply. An approximately 6 week daily supply can contain 40 to 45 unitsof the oral dosage form. An approximately 3 month daily supply cancontain 85 to 95 units of the oral dosage form. An approximatelysix-month daily supply can contain 170 to 200 units of the oral dosageform. An approximately one year daily supply can contain 350 to 380units of the oral dosage form.

A pharmaceutical product containing oral dosage forms for weekly use cancontain 4 or 5 units of the oral dosage form for a monthly supply. Anapproximately 2 month weekly supply can contain 8 or 9 units of the oraldosage form. An approximately 6 week weekly supply can contain about 6units of the oral dosage form. An approximately 3 month weekly supplycan contain 12, 13 or 14 units of the oral dosage form. An approximatelysix-month weekly supply can contain 22 to 30 units of the oral dosageform. An approximately one year weekly supply can contain 45 to 60 unitsof the oral dosage form.

A pharmaceutical product may accommodate other dosing regimes. Forexample, a pharmaceutical product may comprise 5 to 10 units of the oraldosage form, wherein each unit of the oral dosage form contains about 40mg to about 150 mg of zoledronic acid. Some pharmaceutical products maycomprise 1 to 10 units of the oral dosage form, wherein the productcontains about 200 mg to about 2000 mg of zoledronic acid. For such aproduct, each unit of the oral dosage form may be taken daily for 1 to10 days or 5 to 10 days during a month, such as at the beginning of amonth.

Some oral dosage forms comprising zoledronic acid or a salt thereof mayhave enteric coatings or film coatings.

In the examples below, zoledronic acid was used to produce RANK/RANKLantagonism.

EXAMPLES Example 1 Effect of RANK/RANKL Antagonism in Rat Model ofInflammatory Pain

Method:

Inflammatory pain was induced by injection of 100% CFA in a 75 μL volumeinto the left hind paws of SPRAGUE-DAWLEY® rats on day 0, followed byassessments on days 1-4. Animals were orally administered zoledronicacid 3 mg/kg (or 18 mg/m²) to produce RANK/RANKL antagonism, or vehicle(control). Drug was dissolved in distilled water and prepared freshdaily. Animals were fasted prior to dosing.

Values for inflammatory pain (mechanical hyperalgesia) in the vehicleand drug-treated animals were obtained on day 0 prior to CFA injection,and at baseline and post-treatment on days 1-4. Pain was assessed usinga digital Randall-Selitto device (dRS; IITC Life Sciences, WoodlandHills, Calif.). Animals were placed in a restraint sling that suspendedthe animal, leaving the hind limbs available for testing. Pawcompression threshold was measured by applying increasing pressure tothe plantar surface of the hind paw with a dome-shaped tip placedbetween the 3rd and 4th metatarsus. Pressure was applied gradually overapproximately 10 seconds. Measurements were taken from the firstobserved nocifensive behavior of vocalization, struggle or withdrawal. Acut-off value of 300 g was used to prevent injury to the animal.

Reversal of inflammatory pain was calculated according to the formula:% reversal=(Post-treatment−Post-CFA baseline)/(Pre-CFA baseline−Post-CFAbaseline)×100.

Total Pain Relief (TOTPAR), for the 24 hours following vehicle or drugadministration, was calculated as the area under the pain relief(reversal of inflammatory pain) versus time curve, as described in US2014/0107210, using the linear trapezoidal rule. Values for total painrelief were quantified as %·hr, or the product of reversal ofinflammatory pain (%) and time (hr).

The experiment was carried out using 9-10 animals per group.

Results:

RANK/RANKL antagonism with zoledronic acid significantly improvedinflammatory pain thresholds compared to vehicle. Pain thresholdmeasurements taken at various times are shown in FIG. 1. Paw compressionthresholds were higher than for vehicle during the entire measurementperiod after 30 minutes from the start of treatment.

RANK/RANKL antagonism resulted in greater 24-hour Total Pain Relief thanthat achieved with vehicle treatment on all dosing days as shown in FIG.2.

Example 2 Effect of RANK/RANKL Antagonism in Rat Model of Arthritis Pain

Method:

The effect of RANK/RANKL antagonism on arthritis pain was examined inthe rat complete Freund's adjuvant (CFA) model of arthritis pain. Inthis model, injection of 100% complete Freund's adjuvant (CFA) in a 75μL volume into the left hind paws is followed by a 10-14 day period toallow for the development of arthritis pain. Animals were orallyadministered zoledronic acid 9 mg/kg (or 54 mg/m²), or zoledronic acid60 mg/kg (or 360 mg/m²) to produce RANK/RANKL antagonism, divided inthree equal daily doses on the first three days post CFA injection, orvehicle (control). Drug was dissolved in distilled water and preparedfresh daily. Animals were fasted prior to dosing.

Arthritis pain (mechanical hyperalgesia) in the vehicle and drug-treatedanimals was evaluated on day 14 post CFA injection using a digitalRandall-Selitto device (dRS; IITC Life Sciences, Woodland Hills,Calif.). Animals were placed in a restraint sling that suspended theanimal, leaving the hind limbs available for testing. Paw compressionthreshold was measured by applying increasing pressure to the plantarsurface of the hind paw with a dome-shaped tip placed between the 3rdand 4th metatarsus. Pressure was applied gradually over approximately 10seconds. Measurements were taken from the first observed nocifensivebehavior of vocalization, struggle or withdrawal. A cut-off value of 300g was used to prevent injury to the animal.

Reversal of arthritis pain in the ipsilateral (CFA-injected) paw wascalculated according to the formula:% reversal=(ipsilateral drug threshold−ipsilateral vehiclethreshold)/(contralateral vehicle threshold−ipsilateral vehiclethreshold)×100.

The experiment was carried out using 7-10 animals per group.

Results:

RANK/RANKL antagonism with zoledronic acid significantly improvedarthritis pain thresholds compared to vehicle. As shown in FIGS. 3 and4, RANK/RANKL antagonism produced a dose-dependent reversal of arthritispain. A reversal of 33% was observed in the 9 mg/kg group, and reversalof 54% was observed in the 60 mg/kg group.

Example 3 Effect of RANK/RANKL Antagonism in Complex Regional PainSyndrome (CRPS)

The effect of RANK/RANKL antagonism was examined in the rat tibiafracture model of complex regional pain syndrome (CRPS). CRPS wasinduced in the rats by fracturing the right distal tibias of the animalsand casting the fractured hindpaws for 4 weeks, as described in Guo TZet al. (Pain. 2004;108:95-107). This animal model has been shown toreplicate the inciting trauma, natural history, signs, symptoms, andpathologic changes observed in human CRPS patients (Kingery W S et al.,Pain. 2003;104:75-84).

The casts were removed on the 28th day after fracture. Starting on day29, animals were orally administered either zoledronic acid to produceRANK/RANKL antagonism, or vehicle, for 3 weeks. Drug treated animalsreceived zoledronic acid at a dose of 21 mg/kg (126 mg/m²) on the firstday (day 29), followed by 3 mg/kg/day (18 mg/m²/day) thereafter. Drugwas dissolved in distilled water and administered by gavage. Animalswere fasted for 4 hours before and 2 hours after dosing. Bilateraltesting of hindpaw pain was performed at baseline, on day 29 afterfracture, and then weekly for three weeks.

To measure pain (hyperalgesia), an up-down von Frey testing paradigm wasused. Rats were placed in a clear plastic cylinder (20 cm in diameter)with a wire mesh bottom and allowed to acclimate for 15 minutes. The pawwas tested with one of a series of eight von Frey hairs ranging instiffness from 0.41 g to 15.14 g. The von Frey hair was applied againstthe hindpaw plantar skin at approximately midsole, taking care to avoidthe tori pads. The fiber was pushed until it slightly bowed and then itwas jiggled in that position for 6 seconds. Stimuli were presented at aninterval of several seconds. Hindpaw withdrawal from the fiber wasconsidered a positive response. The initial fiber presentation was 2.1 gand the fibers were presented according to the up-down method of Dixonto generate six responses in the immediate vicinity of the 50%threshold. Stimuli were presented at an interval of several seconds.

Reversal of CRPS pain in the fracture hindpaw was calculated accordingto the formula:% reversal=(change in pain threshold from baseline to 4 weekspost-fracture−change in pain threshold from baseline to timepoint aftertreatment start)/(change in pain threshold from baseline to 4 weekspost-fracture)×100.

The experiment was carried out in 6 animals per group. As illustrated inFIG. 5, RANK/RANKL antagonism with zoledronic acid significantlyreversed CRPS pain as compared to vehicle treatment. Three weeks afterdosing RANK/RANKL antagonism resulted in a complete reversal of CRPSpain.

Example 4

PROLIA® (denosumab, 60 mg) is administered subcutaneously to a femalepatient suffering from complex regional pain syndrome. Within 6 monthsafter receiving the treatment, the patient experiences less pain. Theinjection is repeated every six months.

Example 5

PROLIA® (denosumab, 60 mg) is administered subcutaneously to a malepatient suffering from pain associated with arthritis. Within 6 monthsafter receiving the treatment, the patient experiences less pain. Theinjection is repeated every six months.

Example 6

PROLIA® (denosumab, 60 mg) is administered subcutaneously to a malepatient suffering from low back pain. Within 6 months after receivingthe treatment, the patient experiences less pain. The injection isrepeated every six months.

Example 7

PROLIA® (denosumab, 60 mg) is administered subcutaneously to a femalepatient suffering from neuropathic pain. Within 6 months after receivingthe treatment, the patient experiences less pain. The injection isrepeated every six months.

Example 8

PROLIA® (denosumab, 60 mg) is administered subcutaneously to a femalepatient suffering from pain associated with osteoarthritis. Within 6months after receiving the treatment, the patient experiences less pain.The injection is repeated every six months.

Example 9

IMBRUVICA® (ibrutinib, 420 mg taken orally once daily (three 140 mgcapsules once daily)) is administered to a female patient suffering fromcomplex regional pain syndrome. Within 6 months after beginningtreatment, the patient experiences less pain.

Example 10

IMBRUVICA® (ibrutinib, 420 mg taken orally once daily (three 140 mgcapsules once daily)) is administered to a male patient suffering frompain associated with arthritis. Within 6 months after beginningtreatment, the patient experiences less pain.

Example 11

IMBRUVICA® (ibrutinib, 420 mg taken orally once daily (three 140 mgcapsules once daily)) is administered to a male patient suffering fromlow back pain. Within 6 months after beginning treatment, the patientexperiences less pain.

Example 12

IMBRUVICA® (ibrutinib, 420 mg taken orally once daily (three 140 mgcapsules once daily)) is administered to a female patient suffering fromneuropathic pain. Within 6 months after beginning treatment, the patientexperiences less pain.

Example 13

IMBRUVICA® (ibrutinib, 420 mg taken orally once daily (three 140 mgcapsules once daily)) is administered to a female patient suffering frompain associated with osteoarthritis. Within 6 months after beginningtreatment, the patient experiences less pain.

Example 14

IMBRUVICA® (ibrutinib, 560 mg taken orally once daily, (four 140 mgcapsules once daily)) is administered to a female patient suffering fromcomplex regional pain syndrome. Within 6 months after beginningtreatment, the patient experiences less pain.

Example 15

IMBRUVICA® (ibrutinib, 560 mg taken orally once daily, (four 140 mgcapsules once daily)) is administered to a male patient suffering frompain associated with arthritis. Within 6 months after beginningtreatment, the patient experiences less pain.

Example 16

IMBRUVICA® (ibrutinib, 560 mg taken orally once daily, (four 140 mgcapsules once daily)) is administered to a male patient suffering fromlow back pain. Within 6 months after beginning treatment, the patientexperiences less pain.

Example 17

IMBRUVICA® (ibrutinib, 560 mg taken orally once daily, (four 140 mgcapsules once daily)) is administered to a female patient suffering fromneuropathic pain. Within 6 months after beginning treatment, the patientexperiences less pain.

Example 18

IMBRUVICA® (ibrutinib, 560 mg taken orally once daily, (four 140 mgcapsules once daily)) is administered to a female patient suffering frompain associated with osteoarthritis. Within 6 months after beginningtreatment, the patient experiences less pain.

Embodiments

The following are examples of embodiments that are specificallycontemplated by the inventor:

Embodiment 1. A method of treating pain comprising administering apolypeptide, a protein, or a nucleic acid to relieve pain in a mammal inneed thereof, wherein the polypeptide, the protein, or the nucleic acidis a RANK/RANKL antagonist.

Embodiment 2. The method of embodiment 1, wherein the pain is back pain,pain in an extremity, musculoskeletal pain, joint pain, or muscle pain.

Embodiment 3. The method of embodiment 1 or 2, wherein the pain isinflammatory pain, arthritis pain, complex regional pain syndrome, lowback pain, musculoskeletal pain, neuropathic pain, or osteoarthritispain.

Embodiment 4. The method of embodiment 1, 2, or 3, wherein theRANK/RANKL antagonist is OPG (osteoprotegerin) or a variant thereof; ananti-RANKL antibody; a monoclonal anti-RANKL antibody; or a smallinterfering RNA, a microRNA, a precursor molecule, a ribozyme, anantisense nucleic acid, or an aptamer targeting RANKL.

Embodiment 5. The method of embodiment 1, 2, 3, or 4, wherein theRANK/RANKL antagonist is a humanized monoclonal anti-RANKL antibody.

Embodiment 6. The method of embodiment 1, wherein the RANK/RANKLantagonist is denosumab.

Embodiment 7. The method of embodiment 2, wherein the RANK/RANKLantagonist is denosumab.

Embodiment 8. The method of embodiment 3, wherein the RANK/RANKLantagonist is denosumab.

Embodiment 9. The method of embodiment 1, 2, or 3, wherein theRANK/RANKL antagonist is an OPG variant.

Embodiment 10. The method of embodiment 1, 2, or 3, wherein theRANK/RANKL antagonist is a small interfering RNA, a microRNA, aprecursor molecule, a ribozyme, an antisense nucleic acid, or an aptamertargeting RANKL.

Embodiment 11. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or10, further comprising administering a second therapeutic agentindicated for the treatment of pain or another neurological disorder.

Embodiment 12. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11, wherein the mammal does not have cancer.

Embodiment 13. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11, wherein, if the mammal has cancer, a second therapeutic agent isadministered to the mammal for the treatment of cancer, and wherein thesecond therapeutic agent is not a RANK/RANKL antagonist.

Embodiment 14. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13, wherein the mammal is a human being.

Embodiment 15. The method of embodiment 1, wherein the pain ismusculoskeletal pain and the RAN K/RANKL antagonist is denosumab.

Embodiment 16. The method of embodiment 1, wherein the pain is arthritispain and the RANK/RANKL antagonist is denosumab.

Embodiment 17. The method of embodiment 1, wherein the pain is complexregional pain syndrome and the RANK/RANKL antagonist is denosumab.

Embodiment 18. The method of embodiment 1, wherein the pain is low backpain and the RANK/RANKL antagonist is denosumab.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The invention claimed is:
 1. A method of treating or relievingosteoarthritis pain, low back pain, or pain from complex regional painsyndrome in a mammal in need thereof, comprising administering anantibody to the mammal, wherein the antibody: a) is denosumab; or b) hasbinding specificity to RANKL and competes with denosumab for binding toRANKL.
 2. The method of claim 1, wherein the antibody is a monoclonalantibody.
 3. The method of claim 2 wherein the monoclonal antibody is ahuman antibody, a humanized antibody, or a chimeric antibody.
 4. Themethod of claim 1, wherein the antibody has binding specificity toRANKL.
 5. The method of claim 1, wherein about 0.05 mg/kg to about 3mg/kg of the antibody is administered to the mammal being within a fourweek period.
 6. The method of claim 5, where about 0.1 mg/kg to about 20mg/kg of the antibody is administered.
 7. The method of claim 1, whereinthe antibody is administered no more than once a week.
 8. The method ofclaim 1, wherein the antibody is administered daily, weekly, monthly,every three months, every six months, or every year.
 9. The method ofclaim 1, wherein the method is a method of relieving osteoarthritispain.
 10. The method of claim 9, wherein the osteoarthritis affects aknee.
 11. The method of claim 1, wherein the method is a method ofrelieving pain from complex regional pain syndrome.
 12. The method ofclaim 1, wherein the method is a method of relieving low back pain. 13.The method of claim 1, wherein the antibody comprises a) a heavy chaincomprising CDR1, CDR2, and CDR3 of SEQ ID NO: 3; and b) a light chaincomprising CDR1, CDR2, and CDR3 of SEQ ID NO:
 4. 14. The method of claim1, wherein the mammal experiences pain relief within about three monthsafter the antibody is first administered.
 15. The method of claim 1,wherein the antibody comprises a RANKL-binding fragment.
 16. The methodof claim 15, wherein the RANKL-binding fragment is a Fab, a Fab′, aF(ab′)₂, a Fv fragment, a diabody, a linear antibody, or a single-chainantibody molecule.
 17. The method of claim 1, wherein the antibodypartially or fully blocks, inhibits, or neutralizes a biologicalactivity of RANKL.
 18. The method of claim 1, wherein the mammalexperiences pain relief for at least 24 hours.
 19. The method of claim1, wherein the antibody is denosumab.
 20. The method of claim 1, whereinthe antibody has binding specificity to RANKL and competes withdenosumab for binding to RANKL.
 21. The method of claim 1, wherein themammal is a human being.
 22. The method of claim 1, wherein the antibodythat has binding specificity to RANKL and competes with denosumab forbinding to RANKL has at least about 90% sequence identity to denosumab.23. The method of claim 1, wherein the antibody that has bindingspecificity to RANKL and competes with denosumab for binding to RANKLhas at least about 95% sequence identity to denosumab.
 24. The method ofclaim 1, wherein the antibody that has binding specificity to RANKL andcompetes with denosumab for binding to RANKL; has at least about 99%sequence identity to denosumab.